TRITIUM EXCHANGE IN BIOLOGICAL SYSTEMS

,,
COPY AVAILABLE
13fZST
‘1’RITIUM EXCHANGE IN BIOLOGICAL
SYSTEMS
~’. SIRI AND J. ~,%’ERS
UNIVERSITY OF C.\ LIiWRXI.+, BERKfiI.EY, C~L[F-ORXI.L
UXITED ST~TLS OF AMERICA
Abstract -- R&scrm6 –- .iIiHoTat(IIsI --- Resunlen
Tritium exchange in biological studies. ‘W”hene~CT tritium-lalxlled wJtcr is etnployed as a
test solute or tracer in biological systems, an appreci~b!e exchange between trltium and labile t,ydr~
gen atoms cxcurs that frequently a!ltc:s the nature and lntqrretatic,n of exp-riment.~1 rcsuks. The
studies reported hem are concerned u-ith the magnit,xle of the Meet that tcicium exch:iage Lntrc,duces
into measurements ~>ftotal body water and uwrer recta &>lisrn in animals and humans. Direct measure.
ments of exchange were made in rots, guinea pigs, pigeons, and rabbits. Tritium-labelled water
u-as administered intravenously or by nmutir, and tritium spce and turnover determined fr,um the
concentration of tritium in blood. The animals were then desiccated to c{mst, rnt w-eight irr M;!(o
The specific activity of water collected pcriodjcdly during desiccation increased by 50@~ as a result
of isotope effects. Water from combustion of dried rabbit tissues c,mtained about 2°0 of the tritium
.:
originally given to the anim~l. Adipc]se tissue alone contained little or no exchange tritiurn. The : :,:
dried tissues nf the other animals were dehydrated with inactive water and the appearance of tritium
in the v-arer obser}-ed. The specific activity of the water increased in exponential fashion, i. e., I-exp.
(kt), with about 90~0 of exchange c~curring with a half-time r~f 1 h, and the remaining 10~;~ with a . .
half-time of 10 h. ‘I’he total tritium ewrlcted acc.ountd for 1.5 to 3,5~~ of the d,~se given to the animal, ., .
which agrees with the dltference berween the tritium space and t<,tal body \vater determined b? ,’ ,
.’ ‘h
desiccaticm.
An indirect estimate of exchange in humans ~~as derived from cc,ncurrcrrt nw~surwnents of
tritlum and antip}rene spacrs. The average dlffertnce of abcjut 2° ~ in water vollume agrees with
the d[rect estimates of exchanges in animxls
lt is evident that rrltium space should be rcd,lced by about 2° ~ to identify It N ith total body
water. Tbe magnitude and relatively S1OU-rate of exchange nMy also intlucnce the Interpretation
of metaboljc studies with tritlu m.,
Echanges de tritium clans Ies systirues biologiques. Lorsqu’on ut]l)ae de l’cau tritke cc,n,rn’.
solutt IN comme ind[cateur clans lcs s] st~mes b],>lngiques, il sc pr, jduit entrc lC tritiurn et Ies ~tc>llles
Iablles de l’hydrog~ne un 6chmgc ~Pj}rtciable qui mfluc suuvent sur la IMtiore et l}intcrpr~rari,,n
des r&sLIltats de !’exptricnce. [es r!t~dts prisentks ici conccmcnt !’amplcur des et?ctj de cts tchonges
sur la mes..lre de la m.lsse tomle dc I’e~1~de l’organ~smc et Lr d~tcrmin~tion du nicabo[ismc de
I’eau chez l’hornnw et Ies .nimaux, Des mesures de ces tchanges ont &ti pratiquk dwectcment
sur des rafs, des c(lbayes, des pigecms et dcs lapins, .\pr& a.;oir adminis:ri de l’eau triti+e par voic
]ntraveincusc ou buccale, on a ditcrm in< l’cspace et le rcnml\,ellerncnt du tritium d’zrpris la c,m -
centrati ofi de cc dcrnier dam Ie sang I.es anirn~ux cm: M ensu!te desstrh& sous vir!c jusqn>i sr.lhfll.
sation du polds L’acc1vit4 spiclflquc de I’earr pr<lev4c p4r10djqucn1ent au tours de La dessicca:i[>n
s’est accrue de 50°,1 p~r suite d’etftts isot,>pjques, [ ‘ea,u provenant de la c, ~mhustlon de tissus de
lapin des+chis conrcn~it envtr, m 2“ ~ de la dose origjr,ale dc triti.~m adminlstrie, Seuls Ies tiss~,s
ad]peux onr rtvdt peu oil pas du t(Jut d’+ch.~nge de trjtium. I.es t[ssus dcss+chts des aurres zmimaux
ont <[i rehydmt+s avrc de I’cau lnicti~-e et I’on a dtcel+ du tr]tiu[m d,~ns cc:te eau, On 2 constat<
que l’acti~ i[t! sptcliique de [’rclu atlgn>cnta~t scion “CC Ioi exp,,nentic!]e (I -Ck,.) et que 90~ ~ de
I’&change se pmduisait avec une piricde d’une hcurc et les 10° * restfiots zvcc une piriode de dlx
heurcs. I.e rotal du tritium exttait repr&ntwt 1,5 i 3,5(’(, de la dose admlnistrte i i’anilna], ce qui
concc)rde avcc [a dl!T&rence, d$tetnun~e par dessiccati(m, entre l’espace tritit]m et k masse t,xalc
de l’eau de l’organismc
Une estjmaclnn ,rldlrecte dcs &h2nges chcz I’hornme a &t4 Ltabl!e d’apks ks mestitcj d~s esPa~~$
,, 1 .,
,
ye W. SIRI AND J. EVER5
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tritiulm et antipyrine. La difl&ence nmycr-me d’envirun 2°0 relet-~e clans !e volume d’eau concorde
avec les estimations dircctes des &changes chez les mwmaux.
11 est Lvldcnt qu’iI faut di]minuer de 2°0 cn~iron I’espace tritium pour I’identifier avcc !a m?sse
totale de I’eau de l’t,rganisme, L’amplcur et Ie ryti-,me relativement [ent des Manges pcu}-ent 4rgaIe-
mrmt in fluer sut l’interpr<t~tion des <tudcs dc m~t~bolisn]e c~cctutes a I’aide du tritium.
i
Inferc-arnbio deltritio en 10S sistemas bio16gicos. Sielmprc quesecmplea agua tritiacks cc,mo
soluci6n de cnsayo o indicador en sistemas>bio16gicos, se prod,uce entre ICISitomos de tritio y lCIS
I itomos L+biles de hidrr@eno un intcrcambio appreciable que afecta a menudo a la inciole e intec-
prctaci6n de 10S resultzdos experin~cntales, Los estudios descritos en la presente nwmor!a ticnen
pc,r obieto detemninar la msgnitud de] efecto producido pm el intercamb{o de tritio en las deter-
n-, macionesde] agua del c)rganisrno entero y de] rnetabolismo del agua en 10S anilna!es y en 10S seres
humanos. Sehznefcctu:~do n,cdiciones dircctasdelgsadode if)tcrc~[l>blo enrat~s, cc,bayc>s, pal<)mas
y conqos. Se Ies adn~inistr6 px via intra~-cnosa o por \-is bucal sgua nmrcada con tritlo, y se
dctcrminb tLespaci(~tr[L1c, } larcnc,\ aci<,ndti trltLoa palt)rde lacc)nccntracj(jn detrlrlc, en!asat1gre.
.i continuac)6n ins anlfc.~]es se dt-wcaron CII cl \ acic) hasta a!c~nz~r urr peso ccost.lnte. CumIJ ccm-
secuencla de Ios efcctos is,)t{,picos, ~umentb tm un SOO la acriv:dad especifica del agua recog!da
.
peri(i,dlcalncntc durante e] desecado. El agua i>rc)~ed~~tc de la combustjbn de 10S tejldos dewcados
de cortejo ccmtcnia un ‘2°0 del tritio ,)rtgin.drl-,ente adminls~rado al animal. En el tc]ldo adlp(,so,
tomzdo aisladlmente, el inttTcambic, de trit{o fuc escaso o nulo. Lostejldcrs d~wcados de IOS dermis
al:imales fuerw rchld ratados con aguJ irxrctiva, y se okerv6 la aparici6n de tritio en e] agua. I.a
i, ,,
4:-’
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TR1llUM EXCH.%NGE IN BIOLOGIC<4L SYSTEhi S 73
actit-idad espccitlca del agua aument6 en forma exp,>n.cnch], esr,~ es, c+m arregln a II exprcsi6n
1-exp, (kt); un 90” ,J, apr(.)xin~adari,ci.,re, del interc~nlblc, se pr(tiujo cwi un periodn de una h,jm,
mientras que c1 10’~~ rest~ntc se vcri[ic,~ c{:mun p-rioclo de 10 h{~rss F.1 t:;uc rod ei:traid,-, equl~ ,1[](’,
a una car,tidad que oscilaba entre 1,5 y 3,5’ ~ de la dr,sis aclministrsd~ d .{n!ma!, 10 cud cu:lcuerd~
crm 1Adifcrcnc,a ~ntre c1 esi,acl,.l Lrltio Y e] sgu2 contcnidz en c! ,Jrganlsm,] enter, , y dctcrmin:lda
pof desecadc}.
Se efccru{> una ev.du.{ci,~,n indircc[a de! intercamb~~l pr,.~ducdo en sercs hu~,anc,s ut; l,z.u.d, )
medIcI,.,Iws c~.}mhlnad.?s del esy~cic rritl, , y d:l cspac:(] .In[[pirco, ~. La d! ftrtnck med,~ dc un 20,,,
‘aprr,~lm.dfimtmte, m el volu:nen del 2gu.3 c[,ncucrda cim las de[ermrnqci~mcs d!rcctas del icter-
cambjo en anmm! es,
Es evidcnre que cl es~,clo tritic, debe reduclrse cn un 2“ ~, a tin de quc woincidz c,,n el volurnen
de agm del orgwisnw enrtro, La rrmgnitud y la vclwidxd de interc.~whio relati,-antmte baja pucden
intluir tambl,% en la interprctacir’,n de ios es; udios sobcc el metaholismci efectusdos mediante e!
tritio.
—
s
Biological tracer stuc!ies with tritium, and particularly those involving tritiated
water, are potentially s,-tbject to at least three effects, other than outright radi Jtion
dm-nage, that may influence the precision and at times the interpretation of experi-
:,
mental results. ..
The first of these effects, which is the principal subject of this report, results from
tritium ions behaving like hydrogen ions and, therefore, exchanging with !abile
hydrogen atoms of solutes in aqueous solution. In tissue, for example, e}-ery con-
stituent, with the possible exception of neutral fat, quickly acquires a highly labile ,
tag on ~ddition Of tritilted water, either tn-vivo or in-vitro. Con\~ersely, a metab-
o!ite Iabelled in exchangeable hydrogen positions can loose a major portion of its
tritium by exchange before it ever engages in metabolic processes.
A second effect is non-exchangeable Iabelling by metabolic processes and possibly
byother mecharrismsthat are not yetunderstoocl. Metabolic incorporztionof tritium
into cellular constituents in the presence of tritiated water is to be expected in live
tissue, but it may not account wholly for the non-exchangeable Ixbelling if the
observ~tion reported here on pure albumin solution is valid, Possibly labelling
mech~nisrns related to the WILZLL+CH process may be involved [1].
A third factor that must often be considered in tracer sttldie5 with tritiunl is an
isotope effect resulting from the threr-fold greater Cnajs of the t~iron relatiire to
that of the proton. Though frequently negligible, an isotope e,fect may in some
instances ~flect the outcome of a tracer experinlent by a facto: as great as two or
more.
Although hydrogen e~ch~nge, non-exchangeable Iabel[iug, and isotope ef+ects arc
useful in their own right as investigative tools, the work reported in this p~per is
principa]] yconcernec{ v.iththeir nuis~ncev~]ue in bio[ogic~] traccrexperirnents v,-ith
tritium. It should perhaps Ilso be made cle~r at the outset that the study of these
efiects WM not a deliberately planned investigation but one th~t grew somewhat
rmrdomly out of a ~,ariety of studies on met~bolic processes an d water kinetics in
humzns and animals. 1[ is not, therefore, a systern~tic examination of the problem,
but rather Ln estimate of the prob~b!e magnitude of these effects as they may be
encountered when tritiated water is used as a test solute for total body water and
water kinetics.
The methods en~P!oYeL~ in this study involved equipment and procedures th~t arc
in gener~l use and need not be discussed here beyond noting what they were,
Samples of water from biological fluids and tissues were ~ssayed for tritium with
a Tri-Carb liquid scintillation coincidence counter (Packard Instrument Company,
,, A ., ,,
74 W, SIRI AND J. EVERS
%
LJ Grange, Illinois). The liquid scintillator was that formulated by WERBIN, et. d
[2], which consists of 0.3 g POPOP, 12 g PPO, and 125 g naPhthalene/l p-dioxane.
Fifteen ml of scintillator with 0.2 m) water, the usual sample volume, counted with
an efficiency of 17 o/o. This volume of scintillator will support as much as 2.5 ml
water, zlthough the counting efficiency is then reduced by 50 o/o. Samples were
always recounted with an internal standard of tritiated water and appropriate
corrections made for quenching. Water samples from urine,, blood, ~nd tissues were
obtained by vacuum distillation in an appar~tus somewhzt similar to that described
by LINDERSTRCW-LANG [3], which consists of a bent tube with detachable bulbs on
both ends, one of which holds the specimen while the other is immersed in a cold
bath. Driecl tissue s~lnples were first .ombusted in a conventional Pregl appar~tus
and tritium then assayed in the water of co]nbu~tion, In general, errors in counting,
pipetting, weighing, etc., were m~inrained \ve\l below 10/o by suitable prcc~utions.
The influence of hydrogen cxcha:xe on mcawrements of total body water with
hydrogen isotopes w~s known long before tritium became available (or this purpose.
The early ustrs of dcutcriu]m oxide were aw~re that it gove ~n overestimate of
total w~ter, but a reliable value for the correction was never established, .~nd few
investigators were willing to subject their dlta on tots! body water to a necessary
but ill-defined correctic,n. On both rhcorerica 1 and empirical grounds, estimates ~f
hydrogen exchange corrections have ranged from I~to/O to more than 5 O/o of the
total body water indicated by the isotope.
For entirely different purposes, tbc process of hydrogen exchange in pure protein
solutions was carefully cxanlined by LINDERSTRCJM-LANG and I]is associates [3] at
the Carlsberg Laboratories, and the mechanism of exchange is dealt with at length
in other papers to bc found in tbcse proceedings. In general, exchangeable hydrogen
atoms are thc)se bound to oxygen, nitrogen, ~nd strlphur, while bydtqen bound
directly to carbon is considered to be non-exchangeable. Exchange proceeds ex-
ponentially with time in the manner of a first order reaction and presumably with
a characteristic rate const~.nt for each hydrogen posit~on. Hydrogens in end groups
and side chains appear to exchange most rapidly, while those hound to nitrogen
in the backbone of peptide chains undergo relatively slow exchange. Exchange
half-times for a single protein species are observed to range from seconds to as
long as 24 hours, but it is evident that a substantial fraction of the total exchan~e
must occur with half-times in the order of seconds.
A careful studv of exchan~e cur~es for ~ure &bstances is useful in revealinz
features of mole;ular structt;re but serves’little purpose for the intact anima~
otbcr than to show the gross extent of exchange as a function of time after
administration of tritium. “For the pYrPose of arriving at a precise correction for
exchange as a function of time after administration of tritiurn-labelled water,
such a curve would be desirable but extremely difficult to establish. The best
we can hope for at present is a value based on equilibrium conditions, which
may overstate the effect in exoerirnents of \-erv short duration.
Our observations were made’on the reappearance of tritium on dehydration of
dried tissues and whole animals that had been civen tritiated
. water before they
were sacrificed and desiccated. The intery~ls between triti,um administration and
sacrifice r~nged from 1/. to 24 h. Blood san~p!es m-ere taken from the live Jninlal
.
to derermine triti-um space. After desicc~tion to constant weight, the dried tissue
was dehydrated vith inactive mater and frequent samples taken for tritiurn assay
for a period of 2d. Variations in this procedure, which will be noted later, were
Eollowed for selected tissues of mice and rabbits. Two measures of the overzll
f.:,;
p-:,
TRIT[fihi EXCH.\NGE IN B1OLOGIC.%I. SYSTE!.i S 75
magnitude of exch~nge were seer-rred; one from a direct comparison of tritium
space with w2ter volume by desicc~rion, and the other from a direct me.wurerncnt
of tota[ tritium exchanged.
The gross features of exchange observed in these experiments can be sunlmari7ed
in a highly sirnp!ificd formulation, which should be regarded, however, only as a first
order estim~te in a more detailed ~nzlysis. For simplicity, the initial exchange is
regarded m a reversible exchange of h).drogen between P units of tissue and W units
of tritiated water. In the live animal, W’ is iden:ihed ~ith true total body water.
After desiccation of P] units of tissue ~nd rehYdrAticrn with WI units of inactive
water, exch~nge proceeds as before if there has been no subst~ntial alteration in
molecular structure.
Initial exchange Re-exchange
P,?w PI;..’ W’1. (1)
s
The quantity E is defined as g of exchangeable hYclrogen/g of dry tissue, and H
is g of hydrogen~g of water. The quantities C are counts’minig of whatever their
subscripts indicates.
At equilibrium in the original tritiation, the distribution of tritium between w~ter
and cellular materi~l is readily shown to be proportional to the exchange~hle
hydrogen,
-Cv E . . .
== 9E, (2)
Cn = H ,’ -
After desiccati~n and dehydration, precisely the same ratio should be observed if
there are no gross alterations in molecuiar structure. Obviously, this same ratio
should also be found in all subsequent rehydrarions, irrespective of the quclrrtities
of w~ter used at each step. The total exchangeable hydrogen in the whole anim~l,
in selected tissues, or in specific constituents can be obtained in this fashion, alttrou:h
combustion of the dry material for tritium assay and analysis for total hydrogen
are required, which for a whole animal is awkward.
A simpler procedure for estimating total exchznge~ble hydrogen is baled on tbe
specific activities of blood or urine in the live animal ~nd the water of reh Ydr Jtion,
in which case
,
HU’”C”,J
E---- (3)
P, (C,l ~ c,, ,)
In applying E to the specific problem of correcting tritiurn space to true body
water, the fornl of the correction depends upon how E is defined and measure, J
If it is reg~rded as exch~rtgeable hydro~en~g of whole body dry m~<s, then the true
rot~l body w~tcr moy be shown to be
~“ = :It’co -- E.VC ,,
(4)
(H -- E) CII
where VCO is the dose of tritium administered in courrts~min, J{ is the body weigh;,
and Cv is the activity in blood or urine. This formulation is not altogechcr s.~tis-
factory, however, because little or no exchange occurs in depot fat znd bone rnincr~l,
and E is, therefore, depm-ident upon the degree of obesity.
A more rational appro~ch may be made on the basis of exchangeable hydrogen in
lean tissue, The ratio of total protein to total w~ter in most vertebrates appears to
,,,,
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76 W. SIR] AND J. EVERS
t
L
be about 21/72; one can argue for slightly different values, but the differences
introduce only second order uncertainties.
Tke true total body water in a live animal, assuming E has been determined for
lean tissue, is then given by
H1,c#
w’=. (5)
(H -r 0.292 E) C,r
which should, in principle, be applicable to ~11 mammals and relatively free of
dependence on fatness of the animal. Tritium space, which is the volume estirnattd
directly from the activity in Llood, urine, or other fluids, is hosed on simple dilution:
hence,
w:’”-- Vc,,jc u.. (6)
The exch~i]ge error is, therefore, simply 2.6 times the exchangeable h}drogen:
(7)
Before procerdi, ]g to experimental results on exch,lnge, a brief ~nalysis is needed
in explanation of what appears to be non-exchangeable labelling in these and similar
tr~cer studies. If la.lxlling of organic constituents has occurred by metabolic and
other undefined processes, that portion of the l~belling that involves non-exchange-
able hydrogen will persist through repeated desiccation and rehydr~tion. On com-
bustion of dry tissue after it has passed through one or more such stages, the activity
, Cc in the water of conibustion will be the sum of the activity from residual tritium
exchange and llol]-exch.~t~gc.{ble Iabelling, which can be expressed as
C. = (H&C--ECrJ/H (8)
in which H;, is the total hydrogen per unit of dry tissue.
Whatever the true time dependence of non-exchangeable labelling may be, it
aPPears reasonable [O assume that III solutions -with low concentration of tritium,
and for short titnes, labelling proceeds approximately at a constant rate; hence,
RtHpC,JH.
C, z== (9)
R is a constant, in units of reciprocal time, that can be related to the observed
activities in the original tritiated watqr, dehydration water, and that of combustion:
R ==(HpCc—ECt,l)/tHflCr. (lo)
Returning now to experimental results on animals, Fig. 1 illustrates the character
of exchange observed in albumin and desiccated tissues of the rat and mouse. The
extent of exchange is expressed in o/o of the original dose of tritiurn, and for ,tlbumin
it is norm~lized to the protein -warer ratio in !ean tissue, The greater fract~on of
elcll~nge occurs in qn extremely shcort time, but equilibrium is still not attained
at 24 h except in albumin. It is not proposed, howe~er, that t 1 lese cur~-es for rc-
exchan Se represent ~vhat takes place in the live animal. De,lth and desiccation
ueqluewionab]y alter mo!ecular configurations and structure and almost crrtainly
aflects n]~ny of the rjtes of exchanse. The obvious evidence for this is the fact that
,7: .,
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.
Iii::.:.:.:
. ‘.. \ ., : ‘
TRITIIJ?v1 EXCHANGE IN !(T, [C.4L SYSTEM> 77
we have nmer brought an animal b~ck to JrI rehydrxtion. On the othtr hand,
the indicated values for exch~nge at equili n appwr to ‘m? Vl!id and could be
verified by comp~risorr of the calculated t: m spice with total water Obt!iin~L~
From desiccation.
Experiment~l values for non-exch~ngeab!e :Iling wercobtained only inalbunlin
for 48h tri[iation ~nd in mice at 24h. It is c <or from these two points, however,
th~t In some tracer stud]es non-e xchangea ‘@ .~hcl!ing cannot be wholly ignored.
-
.T...-—_
7 —
.—....—
RAT
dA?i GE
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:’,.,,.
& 2- NON-ExCW W.M .
M%SE .1 , >
.,
$
,.
Character of exchange obsewed in albumin and ; ,io;:, tcd tissues of the rar and mous<,
In the experiments with mice it accounts for I -~iw of the total tritium, and equals
200/o of the exchange eflect. For albumirt, m ~-wtchangeable labelling was about
200/o of exchanze labellin~.
Table I sr.rnlm~ri.~es our ~bservations on a YJ.:“etf of anim~ls and tritium-exposure
times. It is immediately evident that somethil:~ \ik<: 2 o/o of the weiSht of dry tissue
is exchangeable hydrogen, which is equivalent ;:, z~:>out 30 o/o of the total hydrogen
. . .
in lean tissue solids. The variations m the vJ!, Im for exchangeable hydrogen are
largely accounted for by differences in the {it ~witent of the tissues an d animals.
It can be seen that lirtle or no cxchan~e OCCI,, in neutral fat. Heart and lung,
. .
wh~ch obtain lrttle fat, are comp~rahle to alb!: [- ~., v,‘hcreas muscle and skin have
significantly lower values because of the prcsc: C< of fat and inclusion of bone in
some muscle samples. These tissues were ag;,ill d:.icd and dehydrated, and within
the limits of experimental error gave v~lues for cl.: hange.lble h~drogen identical to
{
the first. The rabbit organs were not re-ekch,li ,;:g., with inactlvc water but were
com.busted directl} a(ter drying. The pigton pro+~ to be ~ dit+crent kind of anim,ll
in more ways than simply feather. We ha~-e r]o et;, [anatiorl for their e~tremely Io\v
exchangeable hydrogen.
In generll, after z~ h exposure, permanent l~!.wllin: by n)et~bolic and other
processes was fully a fifth as great as that by ~K:hmge. This would account for
. . . . .
about 1.5 o/o of the ln~tlaI tritlum dose piven c}..: ,anlma . ‘1
The exchan~e error in estirrutes of tot;l body w~ttcr with tritium ~re summarized
in Table 11, which includes for comparison an ejtimate of the same error based on
the volume of water from desiccation and ~Pp~rent tritium dilution in the live
anima!. For a variety of reasons, most of them un.~voidabte at the time, the tritium
spaces and hence the corrections based on thcm zrc uncertain within severrt! per-
>.
,, ., !,
78 w’. S:R[ AND J. EVERS
i
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TABLE I
EXCHANGEABLE HYDROGEN AND NON-EXCHANGE.4BLE LABELLING
N-on exchan~slble -,
Range in
Tritiurn g“. Iabdling “
g ‘0 ““’’EX.
hlaterid exposure exchangeable cxch~ngcabk
time h h} dwgen -“<a ngeable
h> drt>gen ,/’
labe!ling
—
.\lbumin .022
Bcnine serum 48 2.0
Mouse (4) 0.5 1.1 1.06--1.11
Ml]use (3) 24 10 0.72--1.11
hf{)use (4) 24
Ileart ~~lung 2.1 1.2--2.9 .22
\fuscle 1.9 .06
Skin -- subc 16 .10
Rat (4) 4 1.5 1.44-1.55
Guinea pig (2) 4 1.1 0.80-1.55
Rabbit (3) 4
I.iver 1.4 1.11--1.70
K{dney 1.2 1.00--1.70
G.1, Trm 1.0 0.90--130
hi uscle 09 0.90-1.30
Pl~slma s[.)l~ds 17 1.66-1.70
Fat 0.0
Pigeon (2) 6 0.8
I
1 cent. Nevertheless. ., thev tend to cormborzte the estimates derived solelv from re-
,
I exchange, which, on the basis of more detailed analysis of the problem, we believe
, to be the more reliable values for exchange error.
I
The ftct that the exchange in mice containing tritiated water for only half an
hour does not differ greatly from that in mice exposed for 24h leads us to believe
. that exchange in-vlvo occurs more r~pidly than is indicated by the exchange curve
for the dehydrated tissues of the mouse. The correction for the guinea pig is low
because of its gross obesity. This would not exPIain the low value for the pigeon,
hotiever. The values formiceancl rats were remarkably uniform among theanima]s
tested, although we have no immediate explanation for subst~ntial differences in
I value between the mouse and rat.
From this preliminary evidence, it would seem that a correction for hydrogen-
tritium exchange depends to some extent on the length of time the animal, or
TABLE II
ERROR IN TC)TAL BODY WATER MEASURED WITH TRITIUM
—
“rrlrlum Error in total body water I
.\n Ima] expnsure : by dcslccation* by re-cxchangc**
time h 0 0
0 0
.. . .
\Iouse (4) 05 3.7 ‘ 4.8
Ifmse (3) .?4 — ~~
Rat (4) 4 6.4 7.1
i Gumca pig (4) 4 1.6 4,2
P]gecm (~)16 25 3,1
( .— —.. ..—
. fTI,r:.,~l,PICC TBM ,,TBU ‘--
(CJ C
. . C.41CLI, f,., r, ,c.C-l hLng,,. d.s+.(.iaj mmal.
.
., ,,
TRITIUM EXCH.+NGE lii SllO1.OC.lC.kL SYSTEMS
T.ABLE 111
ISOTOPE F’R.ACTIOZXATIOF! IN EXPIRED WATER VAPOL’R
V( t. ; -r. t3. \v Sp. act. expired v 3wr
Sex .%ge
. Kg “o u-t. 5P. aC[, urine-& blued
Human F 31 63 56 0,78
M 51 70 54 0.86
F 36 48 66 0.93
M 28 66 67 0.88
,
M ; 63 88 50 0.96
I M I 36 82 49 0.83
~ Pigeon 1 I ~ 0.55
~ Pigecm 2 0.35
—
‘ human, contained the tritiated w~ter, and pe~haps on the anim~l species and dc_gree
of obesity, Although these data suggest tritium exchanges to the extent of about
50/o of the administered dose in m~tnmals, it is obvious that a more detailed
examination is called for.
The question of an isotope effect is one that can be answered only in the context
of the experimental procedure. Obviously, tritium, by virtue of its great mass, will
affect equilibrium constants, distribution coefficients, diffusion rztes, binding, and
..,, . .
even vibrational frequencies. Whether or not alteration of these characteristic con- ,?
‘,;“,,
stmts z!lects the outcome Of a biological tracer experiment depends on the nature
of the process investigated.
In the investigation of total body water and w.lter kinetics with tritium, in which
a quasi-steady state prevails, it can be said aln)ost with fin~lity th~t an isotope efl”ect
does not occur, or at least is immeasurably smlll. Numerorrs investig~tors have
reported no significant differences in the specific activity of tritium in blood, urine,
and other biological fluids once mixing was complete. The same conclusion W&s
arrived at by the author after ass~ying tritium in the blood and urine in some 300
humons ~nd in innumerable anicnlls.
Wtter involved in metabolism ma}-, however, be another mlttcr. It is also clear
that e~pirecf water vapmrr is subjwt to a l~rge znd unmistak, ~hlc isotope effect. This
is strikingly evidtnt in the pigeon, which in the course of exrendccl fllght, stems to
conserve tritium despite rapid ~v~ter turnover. In order to estinla~c the m.~gnitudc
of this effect, pigeons and hunlan subjects wet-c placed in an open circuit respira~ory
system in which dry air W.M inspired, and e~pireci w~tcr vapour W.{S collected in
cold traps. These tests were conciucred some hours or d~ys after administrati~>n of
tritiated wtter to obviate interference from mixing. The specific activity of expired
water vap~ur could then be comp~red with that of blood and urine taken at the
same time. The results of these measurements are sunlmariz,c ‘d in Table III.
In the hum~n subjects, the specific activity of expired water v~pour relative to
that of urine and blood ran~ed from 0.78 to 0.96, ?No obvious p,ltcern of dependence
emerges from these few subjects, and it can only be conclucieti that an isowpe effect
. . . ..
is there and that ]t 1s sugnik ant.
The pigeon, on tbe other hand, is equipped with quite a different respiratory
. .
apparatus and N able to iractlon~te HTC) and H20 with respectable e[ficicnc Y.
Two subjects hardly qualify the data for statistical certification, but with z reduction
in specific activity of 500/o or more, the influence of an isotope effect is unmistak~b!e
in these birds.
,,
4
so W. SIRI AND ]. EVERS
.<
%.
REFERENCES
[1] V711.ZBACH, K. E., j. Amer. (bmr. Sot, 79 (1957) 1013.
[2] \VERBIN, }+, CEI IIKOFF, L 1.., and 13i.\D.3 N. R., P.oc. Sir. exp. Bio!. and .Wf. 102
!% 1 (1959) 8.
[3] LIXDERS’I’R o WL,iNG, K., Symposium on Pi, ,tein Structure, A. Xcuhcrger, cd., John
\Y’ile~- and Sons, Inc., New Yc,rk (1958) 23-34.
DISCUSSION XX XII
p. Sprillgcl] (Australia): I ~ou\d ]ike to elaborate a little on an aspect of hYdro-
gen-tritiurn exchange mcntiorwd briefly in this paper, namely the exchange involving
pure protein. In collaboration with Dr. S. J. Leach in the Division of Protein
Chemistry, CSIKO, Melbourne, we h~ve rccer,t!y undertaken an exchange study on
ribonuclease in tritiated water.
Information reg~rding molecular structure may he obtained by the study of ex-
chan~eable H-atmns in proteins, and Up to now deuterated water has mainly been
used for such investigations. However, tritiated \vater has a number of advantages
and we have employed it with some success. The main advantages may be sunlnm-
rized as follows:
(1) In the case of deoter~ted proteins it is necessary to deuterate as fully as possible
in > 990/0 DZO, which alters the con fornmtion and stability of the original pro-
tein. On the other hand, the sensitivity of tritium detection methods is such that
only tracer amounts of tritium need be employed. We have usually labelled one
atoln/nlole of protein or less, resulting in much less risk of changes in confor-
mation.
(2) In using tritium there are the possibilities of both equilibrium and kinetic isotope
efiects, ‘The former could le~cl to a distribution of tritium between protein solute
and aqueous solvent which is in favour of the soiute, liter-ature values from
such factors in a variety of systems varying between 0.96 and 1.25 (see e. g.
A. R. G. LANG and .S. G. MASON, Card. /. Chern., 38 (1960) 373). However,
. for ribonuclease samples from six different sources, we have found that the
number of exchangeable hydrogen atoms, assuming a distribution factor of
unity, was the theoretical value of 245 + 5. In this instance therefore, the equilib-
riutil isotope effect appears to be absent. Tf)e use of tritiurn instead of deuterium,
however, does lead to a decrease in the observed rates of exchange. This enables
us to follow the initial rates of exchanze in more detail. ‘rl~is finding also casts
some doubt on the interpretation of “slow” and “fast” H-atoms as being, respec- ;
tively H-bonded or not, within the protein. We now think that these numbers arc
in part a reflection of the method of anal) sis and the particular-H-isotope used. 4
j
In the course of our work a numl?ier of new facts have come to !ight regarding ~
the importance of the precious history of the protein in determining the ease with
which all the exchangeable hydrogens are replaced, When the forward-exchange
reaction (ribonuclease + THO) was carried out on commercial sanlpIes of crys-
t.dlized ribonuclease, the incorporation of tritium wm slow and incomplete. This is
in marked contrast to the results for the back-exchange reaction {N, O-tritiated
ribonuclease + HYO) ~vhere exchange was much more rapid.
The rn.~rked difference in resuits obtained betm-emr the t~o procedures is prob-
,-
abl Y a retlectlon ok the difierenccs In pre-treatments of the rlbonuc lease, In the
bac”k-exchange the protein is lyophilized and heated two or three times from
concentrated solution before the exchange reaction is commenced and this may
cause the ICSSaccessible portions of the molecule to be opened up. On the other
...
,
)
;
,,.
f..
*,. -
p’ ,’
+.’.
&,,.,,,.
TRITIL!M EXCH.4NGE [N B1OLOG1CAL SYSTEMS S1
hand, the protein as purchased has presumably had time to refold during several
years of storage, so that the s2mple procedure of dissolution and forward-exchange
is insufficient to make all che H-atoms accessible.
On the practical side I would like to show two slides, one (Fig, 1) of the appa-
—, —... _——-. ——
1
,.
,.,,,
.,’j .,-
:.
,.,
,,:
——.. —.—.—.
Fig. 1
ratus we use for distilling tritiated water off protein solutions. We have found
that by keeping the bath H at -20 ‘C (bath C is at -70 ‘C) further exchange is
minimized during back-exchange as compared to Iyophilizations at room tem-
perature. This is illustrated in Fig. 2 where the initial number of hydrogens ex-
changing is lowered by 15 H atoms.
Q
—-J
3:0 400
For estimation we ha~e generated Ti + Ht by the method of ISBELL and MOYER
[~ RCJ N’u(l 5M7. Sr~~~fl~~5, 63A (1959) 177] with slight modification (Fig. 3). This
:.
,,.
.,,
:,,.. ,.
I
t
I
*
82 W. SIRI AND J. EVERS
f
%.
gave us a calibration curve for two ionization chambers which showed a linear
relationship between ionization between 20 and 500 pc
current and radioactivity
THO, thus showing absence of a measurable isotope effect during gas generation.
Similarly I should mention that during l-HO distillation no measurable isotope
fractionation effects were noticed.
(h
A
Fig. 3
It is hoped to give a more detailed account of our work at the International
Congress of Biochemistry in MOSCOW in August 1961 and to publish the results
in fu\] in the ~ustrdian ]ourrJu~ of chemistry.
With regard to the possibility of tritium exchange in case of C-H bonds, we
have found no evidence of this in our work using model compounds or ribo-
nuclease. Dr. Wilz.bath, answering a question of mine on this subject, also regarded
such a possibility as somewhat remote. I wonder whether the residual tritium
activity in albumin observed by the authors might stem from strongly adsorbed
water. Could Mr, Siri elaborate on how he dried his protein and what was the
specific activity of the TFIO used for the exchange reactions?
he
W. Siri (United States of America): The animal tissues and protein samples that
we dried were first lyophiliz.ed. .kfter nearly complete drying, the temperature was
raised to 40 CC in the vessel, so that drying continued to completion at 40 CC. As
regards C-H bonds, we have no information on the basis of the work we have
done, as to whether or not they are exchangeable. Our conclusions on non-
em-hsngeability in the present case aye based on other work.
K. Wilz.bath [United States of America): I believe that the levels of radiation
in Mr. Siri’s experiment are too )ow to produce any significant amounts of radia-
tion-induced Iabellirrs.. Therefore. Probablv
what he cal!s Wilzbach labellinz is verv. .
either metabolic labeiling or relatively S1OW chemical exchange at activated posi-
tions. I think that the designation radiation Iabelling for these phenomena is a
misnomer. ~
W. Siri: I appreciate Dr. !Vllzbach’s modesty in refusing to accept credit for
this. Perhaps 1 did not indicate strongly enough thtt we were not absolutely certain
thtt it was Wilzbach labelling, Our main reason for so describing it was tbe lack
of a better term but we stand corrected If Dr. Wilzbach feels that the term does
not apply.
.
,, ,,
TRITIUM EXCH.4NGE IN BIOLOG!C.AL SYSTEMS 8:1
J. Varshavsky (Union of Soviet Socialist Republics): We know from the work
of the school of Linderstrom and Lang th~t the rate of hydrogen exchange varies
in the different O-H and N-H bonds of proteins, hfore specifically, it is known
that the hydrogen aton~s of IS-H bonds participating in the formation of hydrogen
bonds have great difficulty in entering into an exchange and behave to a large
extent in a manner similar to the hydrogens of the C-H bonds. The same picture
is found in nucleic acids and other high- molecul~r compounds of living organisms.
I would be interested in knowing whether the authors of the p~per h~ve con-
sidered the places of possible tritiurn introduction into the bonds in the light of
the sharp differences in the rates of exchange for the various hydrogen atoms
and whether, generally speaking, they attempted to go beyond “gross” investiga-
tion to the possibility of interpreting their results in molecular terms.
W. Siri: Let me say first that we were concerned not with the kinetics of the
reaction but rather with its gross effects, @sofar as they invoive the biologist
and the whole organism. With regard to exchangeable hydrogen, the only way
we could differentiate between it and what we -– perhaps n~ively — chose to
call Wilzbach Iabe[ling was this: after 3—-4 desiccations and dehydrations, there ,,,
still remained a residual radioactivity far greater, i.e. by orders of magnitude, ,,..,’
,
.,,
.,
than we could account for by any of the known exchange processes. If this is .f.
,. A
still exchznge, we have no explanation for it. We are not kineticist~ or rnolecu!ar .,.
chemists, so we must leave the question to the experts. I can only say that in the
experiments we have reported on, there was a residual -- and very substantial —
radioactivity in the dried tissues of the animals afte; r“epeated desiccations or
Iyophilizations and dehydrations with inactive water. I find it surprising that
exchange could occur in the initial tritilcion in hydrogen positions and then
remain so firmly bound throughout subsequent desiccations and dehydrations. We
would welcome any inforlnation on this point.
P. R. Schloerb (United States of America): Approxinlatel y0.50/oof adrnini$terecf
isotope water is excreted in the water eacfi hour. This figure is quite uniform and
in a 3—4 hour equilibrium period approach~s the magnitude of the correction
factor described by Mr. Siri. If this excretion factor is omitted, the two errors
would therefore tend to cancel each other. Does the speaker include urine watet-
in the bladder as a part of “total body water”? Shou[d the vari~hle amounts of
water in the gastro-intestinal tract, although re~dily exchangeable, be considered
as tissue water?
W. Siri: You are quite right and this is one of the reasons why estimates of
total body water based on desiccation (i. e. simple measurement of the amount
of the wzter removed from the animal on drying and correction for exchange),
show differences and are not always as reliable as they might appezr to be.
Estimates of this type are confused by such factors as urinary excretion, loss in
weight, high rates of metabolism in small laboratory anim~ls and a vzriety of
other things, including mixing, To obtain what cou!d be considered a fully reliable
estimate of total body warer, it would be necessary to do a complete w~ter balznce,
collecting every bit of water which is lost by evaporation from the lungs, via the
urine and in other ways. This involves a more elabor~te experin~ental procedure
th~n we were able to apply in these experinlcnts, which were concerned w-ith ,;
metabolic problems of body water rather than with the qucvcion of the total
body water, However, I agree that the question of a precise definition of the total
body water and the method by which it is measured is still open. The water
contained in the bladder probably cannot be regarded as a true part of the bod}-
6*, U
T
1
.
.-i
EM .< W. SIRI ANO J. EVk.RS :.,
%
water of the animal but that in the gastrointestinal tract must unquestionably
be so re~ardtd, because it is exchangeable, i.e. the turnover rate in the gut is
relatively This is not true of the bladder, for example, in the human.
fast,
j. Hasan (Finland): Was there any difference between the desiccation times for
mouse and rat ~issue? What was the average time required for desiccation of the
samples which you used in your studies?
W. Siri: We have not been able to observe any significant difference in desiccation
time as between mouse and rat tissue. \!e have foilowed the weight changes of
these tissues very carefully. YOU will recall that the initial part of the desiccation
was done by lyophi]ization. The second part was done in vacuum at 40 ‘C. We
normally continued the desiccation for at least 24 h. There were certainly no
measurable changes in the weight of these tissues after 12 h. In the case of the
whole animal, howe~er, the order of magnitude of desiccation time differs con-
siderably and we conrinued desiccation of the rat for as long as 2 weeks. This was
4-–5 d after the weight had reached a constant value.
J. Hasan: I asked my question brca.trse I was wondering whether an isotope
effect might not be occurring during the desiccation, so that the concentration
of tritium in the samp!e wdter was increasing -with time and simultaneously ex-
changing with the tissues (which Dr. Springe]l has shown to be possible e~-en in
[ the case of a frozen sample). ‘J%is might explain the difference found by Mr. Siri
between the exchange rates of mouse and rat tissues. From the metabolic rates, ,;
a ditlerence in the opposite direction might be expected.
W. Siri: Unquestionably there is such an eflect. I did not show our data on ~
the change of activity in the animal or in the tissues, but I can assure you that
the last portion of water that comes off in the desiccation has about a 20—300/o
higher specific activity than the water that comes off initially. However, I think
that this fact has relati~ely little influence on the experiments I have described,
because the tissues were in fact freeze-dried, It is unlikely under these cir-
cumstances, i. e. the presence of a solid state, that exchange would take place,
even though it took a number of hours to dry the tissues.
i
I
!
!
9